Rheumatoid arthritis (RA) remains a major unmet medical need despite recent advances in therapy. The pain and loss of function resulting from chronic synovitis are due to a variety of influences, including activated adaptive and innate immune responses. In this proposal, we hypothesize that the low pH environment of the joint can exacerbate synovitis by activating acid sensing channels (ASIC) on synoviocytes to increase production of inflammatory mediators. Our prior experiments in the initial application show that deletion of ASIC3, or local blockade of ASICs, prevents the pain behaviors associated with muscle or joint insult. We further show upregulation of ASICs in sensory neurons innervating inflamed tissue, and alterations in ASIC currents after inflammation. We recently discovered that synoviocytes express ASIC3 and preliminary data show that decreases in pH increase intracellular calcium in synoviocytes from ASIC3+/+ mice but not ASIC3-/- mice. Further, decreases in pH enhance prostaglandin E2 production in fibroblast-like synoviocytes (FLS) from ASIC3+/+ mice. The activation of ASICs by decreases in pH could also activate intracellular signaling cascades that regulate production of cytokines implicated in RA, such as IL-1, IL-6, and TNF. These inflammatory mediators enhance the inflammatory process, regulate joint destruction, and sensitize nociceptive joint afferents. Specific Aim 1 will determine if decreases in pH activate murine and human fibroblast-like synoviocytes (FLS) by increasing intracellular calcium, activating intracellular signaling cascades, and enhancing release inflammatory mediators. Specific Aim 2 will determine if the pH-induced changes in intracellular calcium, signaling cascades, and release of inflammatory mediators, depend on activation of ASIC3 by comparing responses in FLS from ASIC3-/- mice to ASIC3+/+ mice (in Specific Aim 1), and by comparing effects of miRNA knockdown of ASIC3 in human FLS to those in human FLS treated with control miRNA. Specific Aim 3 will determine if ASIC3 contributes to development of inflammation and pain in the passive K/BxN serum transfer model of rheumatoid arthritis by comparing responses in ASIC3-/- and ASIC3+/+ mice. Understanding the novel mechanisms of how pH and ASIC3 enhance the inflammatory process will potentially lead to improved healthcare through development of new pharmaceuticals that target these pathways. PUBLIC HEALTH RELEVANCE: Rheumatoid arthritis (RA) remains a major unmet medical need resulting in pain and loss of function due to chronic inflammation. Decreases in pH, that occur in joints of people with rheumatoid arthritis, can activate acid sensing ion channels, located on synoviocytes, to increase the inflammatory process and thus would be ideal targets for the acidic environment of an inflamed joint. Thus, pharmaceutical agents aimed at inhibition of ASICs are promising in that they not only have the potential to reduce the inflammatory process but also have a direct role in inhibiting musculoskeletal pain.